Vaccine using papilloma virus e proteins delivered by viral vector

ABSTRACT

Cell-mediated immune response to a  papillomavirus  infection can be induced by vaccination with DNA encoding  papillomavirus  E genes. E genes can both prevent the occurrence of  papillomavirus  disease, and treat disease states. Canine  papillomavirus  (COPV) E genes which are codon-optimized to enhance expression in host cells are also given.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a vaccine inducing cell-mediated immunitywhich comprises a vector encoding a papillomavirus E gene, and theprevention and/or treatment of disease caused by the papillomavirus.This invention also relates to adenoviral vector constructs carryingcanine papillomavirus (COPV) “E” genes, and to their use as vaccines.Further inventions also relates to various COPV genes which have beencodon-optimized, and to methods of using the adenoviral constructs.

BACKGROUND OF THE INVENTION

Papillomavirus infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, snakes, monkeys and cows.Papillomaviruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection.Papillomaviruses are species specific infective agents; a humanpapillomavirus cannot infect a non-human.

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses what encode up to eight early and two late genes. The openreading frames (ORFs) of the virus are designated E1 to E7 and L1 andL2, where “E” denotes early and “L” denotes late. L1 and L2 code forvirus capsid proteins. The early genes are associated with functionssuch as viral replication and cellular transformation.

In humans, different HPV types cause distinct diseases, ranging frombenign warts (for examples HPV types 1, 2, 3) to highly invasive genitaland anal carcinomas (HPV types 16 and 18). At present there is not asatisfactory therapeutic regimen for these diseases.

In dogs, canine oral papilloma virus (COPV) causes a transitory outbreakof warts in the mouth. In rabbits, cottontail rabbit papilloma virus(CRPV) can cause cornified warty growths on the skin.

Immunological studies in animals (including dogs) have shown that theproduction of neutralizing antibodies to papillomavirus antigensprevents infection with the homologous virus. Furthermore, immunizationof dogs with DNA encoding the L1 capsid protein of COPV inducesneutralizing antibodies and protects dogs from COPV-induced disease. Inrabbits, immunization with DNA encoding CRPV L1 induces neutralizingantibodies that are partially protective against CRPV disease. Also ithas been shown that immunization with DNA encoding CRPV E proteins, canalso partially protect domestic rabbits from the development of warts inthe absence of neutralizing antibodies. [Han, R. et al. 1999a J Virol73(8), 7039-43; Han, R. et al 1999b Vaccine 17(11-12), 1558-66;Sundaram, P. et al 1997 Vaccine 15(6-7), 664-71; Sundaram, P., et al,1998. Vaccine 16(6), 613-23.]

SUMMARY OF THE INVENTION

This invention relates to the induction of cell-mediated immuneresponses by immunization of animals with adenovirus vectors carryinggenes which encode papillomavirus E proteins (regardless of viral type),and to the protection of immunized animals from disease. The disease canbe induced by infection with a papillomavirus or it can be a modeldisease such as protection from tumor outgrowth by cells expressing an Eprotein as a model tumor antigen.

Thus, this invention relates to a method of preventing a disease causedby a papillomavirus comprising the steps of administering to a mammal avaccine vector comprising a papillomavirus E gene. This invention alsorelates to a method of treating a disease caused by a papillomaviruscomprising administering to a mammal exhibiting symptoms of the diseasea vector comprising a papillomavirus E gene. In both of theseinventions, the mammal is preferably a human, and the vector may beeither an adenovirus vector or a plasmid vector, and the genes arepreferably from a human papillomavirus (HPV) serotype which isassociated with a human disease state. The disease may be, for example,cervical carcinoma, genital warts, or any other disease which isassociated with a papillomavirus infection.

In some embodiments of this invention, protection from disease, oralternatively treatment of existing disease is induced by immunizationwith vectors encoding a protein selected from the group consisting of:E1, E2, E4, E5, E6 and E7 proteins, and combinations thereof. The Eproteins which are particularly preferred are E1 and E2 proteins,delivered either separately or in combination. The polynucleotideencoding the E protein is preferable codon-optimized for expression inthe recipient's cells.

In a particularly preferred embodiment, the vector is an adenoviralvector comprising an adenoviral genome with a deletion in the adenovirusE1 region, and an insert in the adenovirus E1 region, wherein the insertcomprises an expression cassette comprising:

-   -   a) a polynucleotide encoding a papillomavirus protein selected        from the group consisting of E1, E2, E4, E5, E6, E7, and        combinations thereof, or mutant forms thereof, wherein the        polynucleotide is codon-optimized for expression in a human host        cell; and    -   b) a promoter operably linked to the polynucleotide. The        preferred adenovirus may be an Ad 5 adenovirus, but other        serotypes may be used, particularly if one is concerned about        interaction between the adenoviral vector and the patients'        preexisting antibodies.

Another type of vector which is envisioned by this invention is ashuttle plasmid vector comprising a plasmid portion and an adenoviralportion, the adenoviral portion comprising: an adenoviral genome with adeletion in the adenovirus E1 region, and an insert in the adenovirus E1region, wherein the insert comprises an expression cassette comprising:

-   -   a) a polynucleotide encoding an E protein selected from the        group consisting of—E1, E2, E4, E5, E6, E7, and combinations        thereof, or mutant forms thereof, wherein the polynucleotide is        codon-optimized for expression in a mammalian host cell; and    -   b) a promoter operably linked to the polynucleotide.

This invention also is directed to plasmid vaccine vectors, whichcomprise a plasmid portion and an expressible cassette comprising

-   -   a) a polynucleotide encoding an E protein selected from the        group consisting of E1, E2, E4, E5, E6, E7 and combinations        thereof, or mutant forms thereof, wherein the polynucleotide is        codon-optimized for expression in a mammalian host cell; and    -   b) a promoter operably linked to the polynucleotide.

Yet another aspect of this invention are host cells containing thesevectors.

This invention also relates to oligonucleotides which encode a canineoral papillomavirus (COPV) protein which have been codon-optimized forefficient expression in a host cell; preferably the oligonucleotides areDNA.

This invention also relates to a method of making a COPV E proteincomprising expressing in a host cell a synthetic polynucleotide encodinga COPV E protein, or mutated form of the COPV E protein which hasreduced protein function as compared to wild-type protein, but whichmaintains immunogenicity, the polynucleotide sequence comprising codonsoptimized for expression in a mammalian host.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the nucleotide sequence of a codon-optimized COPV E1 gene(SEQ.ID.NO:1).

FIG. 2 is the nucleotide sequence of a codon-optimized COPV E2 gene(SEQ.ID.NO:2).

FIG. 3 is the nucleotide sequence of a codon-optimized COPV E4 gene(SEQ.ID.NO:3).

FIG. 4 is the nucleotide sequence of a codon-optimized COPV E7 geneSEQ.ID.NO:4). In this particular sequence, the cysteine residue atposition 24 has been changed to glycine, and the glutamic acid residueat position 26 has been changed to a glycine.

FIG. 5 is a table showing cell-mediated immune responses in miceimmunized with either an E protein or an L protein.

FIG. 6 is a graph showing the protection of mice from HPV E2 tumorchallenge by immunization with Ad-TO-HPV16E2.

FIG. 7 is a table showing specific cellular immune response in Rhesusmacaques following immunization with Ad5-HPV16 constructs

FIG. 8 is a table summarizing the results of immunizing beagles withAd-COPV E vaccines.

SUMMARY OF THE INVENTION

The term “promoter” as used herein refers to a recognition site on a DNAstrand to which the RNA polymerase binds. The promoter forms aninitiation complex with RNA polymerase to initiate and drivetranscriptional activity. The complex can be modified by activatingsequences termed “enhancers” or inhibiting sequences termed “silencers”.

The term “cassette” refers to the sequence of the present inventionwhich contains the nucleic acid sequence which is to be expressed. Thecassette is similar in concept to a cassette tape; each cassette has itsown sequence. Thus by interchanging the cassette, the vector willexpress a different sequence. Because of the restrictions sites at the5′ and 3′ ends, the cassette can be easily inserted, removed or replacedwith another cassette.

The term “vector” refers to some means by which DNA fragments can beintroduced into a host organism or host tissue. There are various typesof vectors including plasmid, virus (including adenovirus),bacteriophages and cosmids.

The term “effective amount” means sufficient vaccine composition isintroduced to produce the adequate levels of the polypeptide, so that animmune response results. One skilled in the art recognizes that thislevel may vary.

“Synthetic” means that the COPV gene has been modified so that itcontains codons which are preferred for mammalian expression. In manycases, the amino acids encoded by the gene remain the same. In someembodiments, the synthetic gene may encode a modified protein.

“Mutant” as used throughout this specification and claims requires thatif referring to a nucleic acid, the protein encoded has at least thesame type of biological function as the wild-type protein, although themutant may have an enhanced or diminished function; or if referring to aprotein, the mutant protein has at least the same type of biologicalfunction as the wild-type protein, although the mutant may have anenhanced or diminished function.

The term “native” means that the gene contains the DNA sequence as foundin occurring in nature. It is a wild type sequence of viral origin.

DETAILED DESCRIPTION OF THE INVENTION

Synthetic DNA molecules encoding various HPV proteins and COPV proteinsare provided. The codons of the synthetic molecules are designed so asto use the codons preferred by the projected host cell, which inpreferred embodiments is a human cell. The synthetic molecules may beused in a recombinant adenovirus vaccine which provides effectiveimmunoprophylaxis against papillomavirus infection through cell-mediatedimmunity.

The recombinant adenovirus vaccine may also be used in variousprime/boost combinations with a plasmid-based polynucleotide vaccine.This invention provides polynucleotides that, when directly introducedinto a vertebrate in vivo, including mammals such as primates, dogs andhumans, induce the expression of encoded proteins within the animal.

The vaccine formulation of this invention may contain a mixture ofrecombinant adenoviruses encoding different HPV type protein genes (forexample, genes from HPV6, 11, 16 and 18), and/or it may also contain amixture of protein genes (i.e. L1, E1, E2, E4 and/or E7). In similarfashion, the vaccine formulation of this invention may contain a mixtureof recombinant adenoviruses, each encoding different a differentpapillomavirus protein gene (for example, L1, E1, E2, E4 and/or E7). E2genes are particularly preferred.

Serotypes of HPV which are useful in the practice of this inventioninclude: HPV6a, HPV6b, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39,HPV45, HPV51, HPV52, HPV56, HPV58, and HPV68.

Codon Optimization

The wild-type sequences for HPV and COPV genes are known. In accordancewith this invention, papillomavirus gene segments were converted tosequences having identical translated amino acid sequences but withalternative codon usage as defined by Lathe, 1985 “SyntheticOligonucleotide Probes Deduced from Amino Acid Sequence Data:Theoretical and Practical Considerations” J. Molec. Biol. 183:1-12,which is hereby incorporated by reference. The methodology may besummarized as follows:

-   -   1. Identify placement of codons for proper open reading frame.    -   2. Compare wild type codon for observed frequency of use by        human genes.    -   3. If codon is not the most commonly employed, replace it with        an optimal codon for high expression in human cells.    -   4. Repeat this procedure until the entire gene segment has been        replaced.    -   5. Inspect new gene sequence for undesired sequences generated        by these codon replacements (e.g., “ATTTA” sequences,        inadvertent creation of intron splice recognition sites,        unwanted restriction enzyme sites, etc.) and substitute codons        that eliminate these sequences.    -   6. Assemble synthetic gene segments and test for high-level        expression in mammalian cells.

These methods were used to create the following synthetic gene segmentsfor various papillomavirus genes by creating a gene comprised entirelyof codons optimized for high level expression. While the above procedureprovides a summary of our methodology for designing codon-optimizedgenes for DNA vaccines, it is understood by one skilled in the art thatsimilar vaccine efficacy or increased expression of genes may beachieved by minor variations in the procedure or by minor variations inthe sequence.

In some embodiments of this invention, alterations have been made(particularly in the E-protein native protein sequences) to reduce oreliminate protein function while preserving immunogenicity. Mutationswhich decrease enzymatic function are known. Certain alterations weremade for purposes of expanding safety margins and/or improvingexpression yield. These modifications are accomplished by a change inthe codon selected to one that is more highly expressed in mammaliancells.

In accordance with this invention, COPV E7, conversion of cysteine atposition 24 to glycine and glutamic acid at position 26 to glycine waspermitted by alteration of TGC and the GAG to GGA and GGC, respectively.For HPV, mutants include HPV 16 E1 where glycine at amino acid 482 ischanged to aspartic acid and tryptophan at 439 is changed to arginine.For HPV16 E2, a mutant changes glutamic acid at position 39 to alanine;for HPV 16 E7, a mutant changes cysteine at position 24 to glycine, andglutamic acid at 26 is changed to glycine.

The codon-optimized genes are then assembled into an expression cassettewhich comprises sequences designed to provide for efficient expressionof the protein in a human cell. The cassette preferably contains thecodon-optimized gene, with related transcriptional and translationscontrol sequences operatively linked to it, such as a promoter, andtermination sequences. In a preferred embodiment, the promoter is thecytomegalovirus promoter with the intron A sequence (CMV-intA), althoughthose skilled in the art will recognize that any of a number of otherknown promoters such as the strong immunoglobulin, or other eukaryoticgene promoters may be used. A preferred transcriptional terminator isthe bovine growth hormone terminator, although other knowntranscriptional terminators may also be used. The combination ofCMVintA-BGH terminator is particularly preferred.

Examples of preferred gene sequences for COPV E1, E2, E4 and mutant E7(C24G, E26G) are given in SEQ.ID.NOS: 1-4.

Vectors

In accordance with this invention, the expression cassette encoding atleast one papillomavirus protein is then inserted into a vector. Thevector is preferably an adenoviral vector, although linear DNA linked toa promoter, or other vectors, such as adeno-associated virus or amodified vaccinia virus vector may also be used.

If the vector chosen is an adenovirus, it is preferred that the vectorbe a so-called first-generation adenoviral vector. These adenoviralvectors are characterized by having a non-functional E1 gene region, andpreferably a deleted adenoviral E1 gene region. In some embodiments, theexpression cassette is inserted in the position where the adenoviral E1gene is normally located. In addition, these vectors optionally have anon-functional or deleted E3 region. The adenoviruses can be multipliedin known cell lines which express the viral E1 gene, such as 293 cells,or PERC.6 cells, or in cell lines derived from 293 or PERC.6 cell whichare transiently or stablily transformed to express an extra protein. Forexamples, when using constructs that have a controlled gene expression,such as a tetracycline regulatable promoter system, the cell line mayexpress components involved in the regulatory system. One example ofsuch a cell line is T-Rex-293; others are known in the art.

For convenience in manipulating the adenoviral vector, the adenovirusmay be in a shuttle plasmid form. This invention is also directed to ashuttle plasmid vector which comprises a plasmid portion and anadenovirus portion, the adenovirus portion comprising an adenoviralgenome which has a deleted E1 and optional E3 deletion, and has aninserted expression cassette comprising at least one codon-optimizedpapillomavirus gene. In preferred embodiments, there is a restrictionsite flanking the adenoviral portion of the plasmid so that theadenoviral vector can easily be removed. The shuttle plasmid may bereplicated in prokaryotic cells or eukaryotic cells.

Standard techniques of molecular biology for preparing and purifying DNAconstructs enable the preparation of the adenoviruses, shuttle plasmidsand DNA immunogens of this invention.

In some embodiment of this invention, both the adenoviral vectorsvaccine and a plasmid vaccine may be administered to a vertebrate inorder to induce an immune response. In this case, the two vectors areadministered in a “prime and boost” regimen. For example the first typeof vector is administered, then after a predetermined amount of time,for example, 1 month, 2 months, six months, or other appropriateinterval, a second type of vector is administered. Preferably thevectors carry expression cassettes encoding the same polynucleotide orcombination of polynucleotides. In the embodiment where a plasmid DNA isalso used, it is preferred that the vector contain one or more promotersrecognized by mammalian or insect cells. In a preferred embodiment, theplasmid would contain a strong promoter such as, but not limited to, theCMV promoter. The gene to be expressed would be linked to such apromoter. An example of such a plasmid would be the mammalian expressionplasmid V1Jns as described (J. Shiver et. al. 1996, in DNA Vaccines,eds., M. Liu, et al. N.Y. Acad. Sci., N.Y., 772:198-208 and is hereinincorporated by reference).

Thus, another aspect of this invention is a method for inducing animmune response against a papillomavirus in a mammal, comprising

-   -   A) introducing into the mammal a first vector comprising a        polynucleotide encoding a papillomavirus protein selected from        the groups consisting of E1, E2, E4, E6, E7, combinations        thereof, and mutants thereof;    -   B) allowing a predetermined amount of time to pass;    -   C) introducing into the mammal a second vector comprising an        adenoviral genome with a deletion in the E1 region, and an        insert in the E1 region, wherein the insert comprises an        expression cassette comprising:    -   i) a polynucleotide encoding an COPV protein selected from the        group consisting of, E1, E2, E4, and E7 proteins, combinations        thereof, and mutant forms thereof; and    -   ii) a promoter operably linked to the polynucleotide.

In some embodiments, the first vector be a plasmid vaccine vector andthe second vector be an adenoviral vector.

In yet another embodiment of this invention, the codon-optimized genesare introduced into the recipient by way of a plasmid or adenoviralvector, as a “priming dose”, and then a “boost” is accomplished byintroducing into the recipient a polypeptide or protein which isessentially the same as that which is encoded by the codon-optimizedgene. Fragments of a full length protein may be substituted, especiallythose with are immunogenic and/or include an epitope.

It is also a part of this invention to combine the use of the nucleotidebased vaccines with the administration of a protein. The protein may bean L1 protein, or an L1 in combination with an L2 protein. It isparticularly preferred that the protein be in the form of a VLP. The VLPmay be a human papillomavirus VLP. Such VLPs are known and described inthe art.

The amount of expressible DNA or transcribed RNA to be introduced into avaccine recipient will depend partially on the strength of the promotersused and on the immunogenicity of the expressed gene product. Ingeneral, an immunologically or prophylactically effective dose of about1 ng to 100 mg, and preferably about 10%1 g to 300 μg of a plasmidvaccine vector is administered directly into muscle tissue. An effectivedose for recombinant adenovirus is approximately 10⁶-10¹² particles andpreferably about 10⁷-10¹¹ particles. Subcutaneous injection, intradermalintroduction, impression though the skin, and other modes ofadministration such as intraperitoneal, intravenous, or inhalationdelivery are also contemplated. It is also contemplated that boostervaccinations may be provided. Parentaeral administration, such asintravenous, intramuscular, subcutaneous or other means ofadministration with adjuvants such as interleukin 12 protein,concurrently with or subsequent to parenteral introduction of thevaccine of this invention is also advantageous.

The vaccine vectors of this invention may be naked, i.e., unassociatedwith any proteins, adjuvants or other agents which impact on therecipient's immune system. In this case, it is desirable for the vaccinevectors to be in a physiologically acceptable solution, such as, but notlimited to, sterile saline or sterile buffered saline. Alternatively,the DNA may be associated with an adjuvant known in the art to boostimmune responses, such as a protein or other carrier. Agents whichassist in the cellular uptake of DNA, such as, but not limited tocalcium ion, may also be used to advantage. These agents are generallyreferred to as transfection facilitating reagents and pharmaceuticallyacceptable carriers.

The following examples are offered by way of illustration and are notintended to limit the invention in any manner.

EXAMPLE 1

Synthetic Gene Construction

The construction of synthetic codon-optimized gene sequences for humanpapillomavirus type 16 proteins L1, E1, and E2 was disclosed previously(International Publication Number WO 01/14416A2, publication date: 1Mar. 2001, “Synthetic Human Papillomavirus Genes” which is herebyincorporated by reference). Synthetic gene sequences for canine oralpapillomavirus proteins E1, E2, and E7 were generated by reversetranslation of amino acid sequences using the most frequently usedcodons found in highly expressed mammalian genes. (R. Lathe, 1985, J.Mol. Biol. 183:1-12, which is hereby incorporated by reference). Someadjustments to these codon-optimized sequences were made to introduce orremove restriction sites.

Oligonucleotides based on these sequences were chemically synthesized(Midland Certified Reagents; Midland, Tex.) and assembled by PCRamplification. (J. Haas et. al., 1996, Current Biology 6:315-324; andPCR Protocols, M. Innis, et al, eds., Academic Press, 1990, both ofwhich are hereby incorporated by reference).

Full-length sequences were cloned into the mammalian expression vectorV1Jns (J. Shiver et. al. 1996, in DNA Vaccines, eds., M. Liu, et al.N.Y. Acad. Sci., N.Y., 772:198-208, which is hereby incorporated byreference) and sequenced by standard methodology. In cases where theactual sequence differed from the expected and resulted in amino acidsubstitution, that sequence was corrected by PCR mutagenesis aspreviously described (PCR Protocols, M. Innis, et al, eds., AcademicPress, 1990, pg 177-180).

Protein expression was evaluated by transient transfection of equalquantities of plasmid DNA into 293 (transformed embryonic human kidney)cells or C33a cells which were harvested at 48 hr post DNA addition.Cell lysates were normalized to provide equal protein loadings. Analysiswas by immunoblot (Western) analysis using sera prepared to each of theCOPV proteins. (Current Protocols in Molecular Biology, eds., F.Ausabel, et. Al., John Wiley and Sons, 1998, which is herebyincorporated by reference).

EXAMPLE 2

Synthesis of COPV E1

The gene encoding COPV E1 was prepared by the annealing and extension of24 oligomers (83-108 bp in length) designed to encode the final desiredsequence. The oligomers were alternating, overlapping sense andantisense sequences which spanned the entire length of the optimizedCOPV E1 coding sequence as well as providing the following importantsequence elements: (1) BglII and EcoRV restriction sites plus a CCACC“Kozak sequence” upstream of the ATG initiation codon and (2) EcoRV andBglII restriction sites downstream of the translation termination codonat the extreme 5′ and 3′ ends of the synthetic full-length sequence.Each oligomer had a complementary overlap region of 23-27 bp with theadjoining oligomer (duplex had Tm of 78-86° C.). Six separate extensionreactions were performed using four adjoining, overlapping oligomers andsense and antisense PCR primers (20-25 nt in length, Tm=68-70° C.)complementary to the distal 5′ and 3′ portions of the first and fourtholigomer, respectively. The actual conditions of PCR were similar tothose described in EXAMPLES 3 and 4 of International Publication NumberWO 01/14416A2.

As a result of these PCR reactions, the following six fragments of thegene were created: COPV E1-A, COPV E1-B, COPV E1-C, COPV E1-D, COPV E1-Eand COPV E1-F.

The above fragments resulting from the PCR reactions were gel separatedon low melting point agarose with the appropriately-sized productsexcised and purified using the Agarase™ method (Boehringer MannheimBiochemicals) as recommended by the manufacturer. Fragments COPV E1-A,COPV E1-B and COPV E1-C were combined in a subsequent PCR reaction usingappropriate distal sense and antisense PCR oligomers as describedpreviously (International Publication Number WO 01/14416A2), yieldingthe PCR product COPV E1-G. In a similar manner, fragments COPV E1-D,COPV E1-E and COPV E1-F were assembled in a subsequent PCR reaction withthe appropriate primers to yield the fragment COPV E1-H. The completegene was then assembled by an additional PCR reaction in which fragmentsCOPV E1-G and COPV E1-H were combined using appropriate distal sense andantisense PCR primers. The resulting 1.8 kb product (designated COPVE1-I) was gel isolated, digested with Bgl II and subcloned into theexpression vector V1Jns and a number of independent isolates weresequenced. In instances where a mutation was observed, it was correctedby assembling overlapping portions of COPV E1 gene segments fromdifferent isolates that had the correct sequence.

Standard PCR methods as described above were used. DNA was isolated froma final clone with the correct COPV E1 DNA sequence and properorientation within V1Jns for use in transient transfection assays asdescribed in EXAMPLE 1. The sequence of the codon-optimized ORF for COPVE1 is shown in FIG. 1 (SEQ.ID.NO.:1).

Immunoblot analyses of cell lysates prepared from the transfected cellsverified the expression of a protein of the expected size which reactedwith antibodies directed against COPV E1 (results not shown).

EXAMPLE 3

Synthesis of COPV E2, COPV E4 and COPV E7 Genes

The synthetic genes encoding the codon-optimized versions of the COPVE2, COPV°E4 and COPV E7 proteins were prepared using the same type ofconstruction strategy using annealing and extension of long DNAoligomers as described in Example 2 and in International PublicationNumber WO 01/14416A2. The sequences used for the long DNA oligomers andPCR primers used for assembly of the oligomers and resulting genefragments were designed according to the criteria in Example 2 in orderto give the following final coding sequences: COPV E2, FIG. 2(SEQ.ID.NO.:2); COPV E4, FIG. 3 (SEQ.ID.NO.:3).

The codon-optimized COPV E7 gene was initially constructed to encode thewild-type COPV E7 protein sequence. The double mutant (C24G, E26G)version of COPV E7 was prepared by PCR mutagenesis by converting TGC atcodon 24 to GGA and by converting GAG at codon 26 to GGC. The methodsfor the PCR mutagenesis were as previously described (PCR Protocols, M.Innis, et al, eds., Academic Press, 1990, pg 177-180). The final codingsequence used for COPV E7 (C24G,E26G) is shown in FIG. 4 (SEQ.ID.NO.:4).

For all three of these synthetic genes, the following sequence elementswere also present in the final assembled gene fragment in addition tothe protein coding sequence: (1) BglII and PmlI restriction sites plus aCCACC “Kozak sequence” upstream of the ATG initiation codon and (2) PmlIand BglII restriction sites downstream of the translation terminationcodon. As described above for COPV E1, each of the three gene fragmentswas digested with BglII and cloned into the expression vector V1Jns.Following verification of the DNA sequences, purified plasmid DNAs foreach of the three constructs were used for transient transfection assaysas described in Example 1.

For COPV E2, COPV E4 and COPV E7, immunoblot analyses of cell lysatesprepared from the cells transfected with the corresponding vectorverified the expression of a protein of the expected size which reactedwith antibodies directed against that particular COPV protein (resultsnot shown).

EXAMPLE 4

Construction of Replication-Defective Adenovirus Expressing HPV or COPVAntigens

Shuttle vector pHCMVIBGHpA1 contains Ad5 sequences from bp1 to bp 341and bp 3534 to bp 5798 with a expression cassette containing humancytomegalovirus (HCMV) promoter plus intron A and bovine growth hormonepolyadenylation signal.

The adenoviral backbone vector pAdE1-E3—(also named as pHVad1) containsall Ad5 sequences except those nucleotides encompassing the E1 and E3region.

Construction of Ad5-HPV16E1: The HPV16 E1 coding sequence was excisedfrom V1Jns-HPV16E1 by digestion with BglII and cloned into the BglIIsite located between the CMV promoter and BGH terminator inpHCMVIBGHpA1. The resulting shuttle vector was recombined with theadenovirus backbone vector DNA as described previously (InternationalPublication Number WO 01/14416A2). The resulting recombinant virus,Ad5-HPV16E1, was then isolated and amplified in 293 cells as describedin that same reference.

Construction of Ad5-TO-HPV16L1:

Construction of Adenoviral Shuttle Plasmid pA1-TO-HPV16L1 ContainingHPV16L1 Under Control of the Regulated CMV-TO Promoter.

The construction of the plasmid HPV16L1/V1Jns, which contains thecodon-optimized synthetic coding sequence for HPV16L1 was describedpreviously (International Publication Number WO 01/14416A2, publicationdate: 1 Mar. 2001, Synthetic Human Papillomavirus Genes). The syntheticHPV16L1 coding sequence was excised from HPV16L1/V1Jns by digestion withBglII plus EcoRI and then cloned into BglII, EcoRI-digested pHCMVIBGHpA1to yield the shuttle vector pA1-CMVI-HPV16L1. The shuttle vectorpA1-CMVI-HPV16L1 was digested with BglII plus SpeI (to remove the CMVpromoter plus intron A sequences), made flushended and the large vectorfragment was gel-purified.

The mammalian expression vector pcDNA4/TO (Invitrogen Corp.) containstwo copies of the tetracycline operator (TetO₂) sequence inserted 10 bpdownstream of the TATA box sequence for the human CMV promoter presentin that vector. Presence of the tetracycline operator (TetO₂) sequenceresults in repression of expression in host cells that express theTetracycline repressor. The pcDNA4/TO vector was digested with NruI plusEcoRV and the 823 bp fragment bearing the CMV promoter plus tetracyclineoperator (2×TetO₂) sequences (CMV-TO) was gel-purified and ligated withthe aforementioned 8.3 kbp BglII-SpeI (flushended) fragment bearing theHPV16L1 coding sequence. The resulting plasmid was designatedpA1-TO-HPV16L1.

Homoloogus Recombination to Generate Shuttle Plasmid Form of RecombinantAdenoviral Vector pAd-TO-HPV16L1.

Shuttle plasmid pA1-TO-HPV16L1 was digested with restriction enzymesSspI and BstZ17I and then co-transformed into E. coli strain BJ5183 withlinearized (ClaI-digested) adenoviral backbone plasmid pAdE1-E3-. Eightcolonies were picked from the resulting transformation plate andseparately grown in 2-ml of Terrific Broth containing 50 mcg/ml ofampicillin. Small-scale plasmid DNA preparation were made and then usedfor transformation of E. coli STBL2 competent cells (Life Technologies).From each of the resulting transformation plates, a single colony waspicked and inoculated into LB with ampicillin (50 mcg/ml) and grownovernight at 37° C. Plasmid DNA was prepared from each culture andrestriction enzyme analysis was used to verify that the pAd5-TO-HPV16L1plasmids had the correct structure.

Generation of Recombinant Adenovirus Ad5-TO-HPV16L1 in T-REx-293 cells

The shuttle plasmid pAd-TO-HPV16L1 was linearized by digestion with therestriction enzyme PacI and then transfected into T-REx-293 cells (whichexpress the Tetracycline repressor) using the CaPO₄ method (InVitrogenkit). Ten days later, 10 plaques were picked and grown in T-REx-293cells in 35-mm plates. PCR analysis of the adenoviral DNA indicated thatthe virus were positive for HPV16L1.

Evaluation of Large Scale Adenovirus Ad5-TO-HPV16L1

A selected clone was grown into large quantities through multiple roundsof amplification in T-REx-293 cells. Viral DNA was extracted andconfirmed by PCR and restriction enzyme analysis. Expression of HPV16L1was verified by immunoblot analysis of 293 cells infected with therecombinant adenovirus. (Expression from the CMV-TO promoter isdepressed in 293 cells, which do not express the Tetracyclinerepressor).

Construction of Ad5-TO-HPV16E2.

The construction of V1Jns-HPV16E2 containing the codon-optimized HPV16E2coding sequence was described previously (WO 01/14416A2). The codingsequence for HPV16E2 was excised from V1Jns-HPV16E2 by digestion withBglII and the fragment was made flushended. The aforementioned shuttlevector pA1-TO-HPV16L1 was digested with BamHI plus EcoRV to remove theHPV16L1 coding sequence. The resulting vector fragment (pA1-TO) was thenmade flush-ended by treatment with Klenow DNA polymerase and ligatedwith the HPV16E2 DNA fragment, yielding the shuttle vectorpA1-TO-HPV16E2. This latter shuttle vector was digested with restrictionenzymes SgrAI and BstZ17I and then co-transformed into E. coli strainBJ5183 with linearized (ClaI-digested) adenoviral backbone plasmidpAdE1-E3-. The resulting transformants were screened and recombinantAd5-TO-HPV16E2 virus was rescued and expanded in T-REx-293 cells asdescribed above. Expression of HPV16E2 was verified by immunoblotanalysis of 293 cells infected with the recombinant adenovirus.

Construction of Ad5-COPVE1: The coding sequence for COPV E1 was excisedfrom V1Jns-COPV-E1 by digestion with EcoRV and ligated with theaforementioned shuttle EcoRV-BamHI(flushended) pA1-TO vector fragment.,yielding the shuttle vector pA1-TO-COPV-E1. This shuttle vector was thendigested with SgrAI plus BstZ17I and co-transfected into E. coli strainBJ5183 with linearized (ClaI-digested) adenovirus vector backbonepAdE1-E3. The resulting transformants were screened and recombinantadenovirus, Ad5-COPVE1, was then rescued and amplified in T-Rex-293cells as described above. Expression of COPVE1 was verified byimmunoblot analysis of 293 cells infected with the recombinantadenovirus.

Construction of Ad5-COPVE2:: The coding sequence for COPV E2 was excisedfrom V1Jns-COPV-E2 by digestion with PmlI and ligated with theaforementioned EcoRV-BamHI(flushended) pA1-TO vector fragment, yieldingthe shuttle vector pA1-TO-COPV-E2. This shuttle vector was then digestedwith SspI plus BstZ17I and co-transformed into E. coli strain BJ5183with linearized (ClaI-digested) adenovirus vector backbone pAdE1-E3—DNAas described above. Eight single colonies were picked from the resultingtransformation plate and inoculated into 2-ml of Terrific Broth withampicillin (50 mcg/ml) and then grown for 8 hours at 37° C. Cells wereharvested and small-scale plasmid DNA preparations were made(pAd-TO-COPV-E2 isolates). The plasmid DNAs for pAd-TO-COPV-E2 clones#1, 3, 5 and 7 were then transformed into E. coli STBL2 competent cells.Two colonies for each original DNA (colonies 1-1, 1-2, 3-1, 3-2, 5-1,5-2, 7-1 and 7-2) were picked and grown separately in LB with ampicillin(50 mcg/ml) overnight at 37° C. Large-scale plasmid DNA preparationswere then made for pAd-TO-COPV-E2 isolates #7-1 and #7-2. Both purifiedDNAs were digested with HindIII and XhoI to confirm that they had thecorrect structure. Both pAd-TO-COPV-E2 isolates #7-1 and #7-2 weredigested with PacI and transfected into T-REx-293 cells using GTSGeneporter transfection reagent. Six days later, several plaques werepicked and grown in T-REx-293 cells in 35 mm plates. Based on PCRanalysis of the adenoviral DNA, clone #7.1B of Ad-TO-COPV-E2 wasselected for further evaluation. This isolate was grown into largequantities through multiple rounds of amplification in T-REx-293 cells.The virus was then purified by banding on CsCl equilibrium densitygradients. This virus preparation was designated Ad5-COPVE2, ID#7.1 p7.Viral DNA was purified and the structure was confirmed by digestion withthe restriction enzymes HindIII and XhoI. Expression of COPV E2 wasverified by immunoblot analysis of 293 cells infected with therecombinant Ad5-COPVE2 adenovirus.

Construction of Ad5-COPVE4 and Ad5-COPVE7: The coding sequences for COPVE4 and COPV E7 (C24G, E26G double mutant) were excised fromV1Jns-COPV-E4 and V1Jns-COPV-E7, respectively, by digestion with PmlI.The gene fragments were ligated with the aforementionedEcoRV-BamHI(flushended) pA1-TO vector fragment, yielding the shuttlevectors pA1-TO-COPV-E4 and pA1-TO-COPV-E7, respectively. The subsequentsteps of recombination with the pAdE1-E3—vector backbone and the rescueand amplification of the resulting recombinant Ad5-COPVE4 and Ad5-COPVE7viruses in T-REx-293 cells were as described above. Expression of COPVE4 and COPV E7 was verified by immunoblot analyses of 293 cells infectedwith the corresponding recombinant adenovirus.

EXAMPLE 5

Generation of HPV-Specific Cellular Immune Responses in Mice byImmunization with Ad-TO-HPV16E2 or Ad-TO-HPV16L1

Groups of female BALB/c mice were immunized by intramuscular injectionwith 10⁹ virus particles (vp) Ad-TO-HPV16E2 or with 10⁹ vp Ad-TO-HPV16L1(control) at day 0 and day 21. On day 34, two mice from eachimmunization group were randomly chosen, sacrificed, and ELISPOTanalysis was performed on splenocytes. The results are shown in FIG. 5.Animals immunized with Ad-TO-HPV16E2 had developed only HPV 16E2-specific responses, while the Ad-TO-HPV16L1-immunized animalsdeveloped only HPV 16 L1-specific responses.

EXAMPLE 6

IFN-γ ELISpot Assay

Mouse splenocytes were prepared from freshly macerated spleens.Depletion of CD4+ cells was achieved by magnetic bead separation usingDynabeads CD4 (L3T4) (Dynal, Oslo). Briefly, 96-well polyvinylidinedifluoride (PVDF)-backed plates (MAIP NOB 10; Millipore, Bedford, Mass.)were coated with 10 μg anti-murine rIFN-γ (BD PharMingen) per well in100 μl of PBS at 4° C. for 16-20 hours. Plates were washed three timeswith PBS, and then blocked with RPMI-1640 medium containing 10%heat-inactivated FBS. Cells were cultured at 5×10⁵ per well in 0.1 mL ofmedium for restimulation with pools of 20mer peptides comprising theentire amino acid sequence of HPV16 E2, or L1 or matching DMSOconcentration in media as a negative control.

Alternatively, cells were co-cultured with 10⁴ CT26 cells, afully-transformed, tumorigenic syngeneic line, or with 10⁴ JCL031 cells,a clonal isolate derived from CT26 cells that had been transformed toexpress HPV 16 E2 protein. After 20-24 hr incubation at 37° C., theplates were washed 6 times with PBS containing 0.005% Tween 20. Plateswere then incubated with 1 μg biotinylated anti-murine rIFN-γ (BDPharMingen) per well in 50 μl of PBS-Tween+5% FCS at 4° C. for 16-20hours. The plates were washed 6 times with PBS-Tween before the additionof 100 μl per well of Streptavidin-AP conjugate (BD PharMingen), diluted1:2000 in PBS-Tween+5% FCS. After 3 washes with PBS-Tween and 3 washeswith PBS, spots were developed with one-step NBT/BCIP reagent (Pierce,Rockford, Ill.). Spots were counted using an automated detection system.

EXAMPLE 7

Protection of Mice from an HPV E2 Tumor Challenge by Immunization withAd-TO-HPV16E2

Groups of BALB/c mice were immunized by intramuscular injection with 10⁹vp Ad-TO-HPV16E2 or with 10⁹ vp Ad-TO-HPV16L1 (control) at day 0 and day21. On day 43, each group of 18 mice were challenged by s.c. inoculationwith 7.5×10⁵ JCL031 cells, a fully-transformed tumorigenic, isogeniccell line that expresses HPV16 E2 derived from the CT26 cell line.

Briefly, the plasmid, pBJ-16 E2, which induces E2 protein expression intransiently-transfected A293 or CT26 cells, was transfected into CT26cells using Lipofectamine (Gibco BRL, Gaithersburg, Md.). CT26 cells, afully-transformed line derived from a BALB/c mouse colon carcinoma, havebeen widely used to present model tumor antigens. (Brattain et al., 1980Cancer Research 40:2142-2146; Fearon, E. et al., 1988 Cancer Research,48:2975-2980; both of which are incorporated by reference). After two tothree weeks growth in selective medium containing 400 μg/mL G418,well-isolated colonies of cells were recovered using cloning rings andtransferred to 48-well plates. One clone was positive for E2 expressionby immunoblot analysis and was subjected to two further rounds ofcloning by limiting dilution. One G418 resistant, E2-positive clonalisolate was used to established the cell line JCL-031.

Animals were monitored for tumor outgrowth for four weeks. The resultsare shown in FIG. 2. Animals immunized with the Ad-TO-HPV16E2 virus werewell-protected from tumor out-growth; 17 of 18 remained tumor-freeduring the observation period. In the control group, 16 of 18 micedeveloped tumors.

EXAMPLE 8

Generation of HPV16-Specific Cellular Immune Responses in RhesusMacaques by Immunization with Ad5 HPV-16 Constructs

Cohorts of 3 or 4 Rhesus macaques were vaccinated intramuscularly atweeks 0 and 24 with 10¹¹ Ad5-TO-HPV16L1, Ad5 HPV16-E1, or Ad5 HPV16-L2virus particles. PBMC samples were collected at selected time points andassayed for antigen-specific IFN-γ secretion following overnightstimulation with HPV16 L1, E1, or E2 20mer peptide pools via ELISpotassay.

The results shown in FIG. 7 demonstrate a strong cellular immuneresponse to HPV16 L1, E1, and E2 following a single dose of the Ad5HPV16 constructs. These data also demonstrate that the cellularresponses can be boosted by vaccination with a second dose of the Ad5HPV16 constructs.

EXAMPLE 9

IFN-γ ELISpot Assay

Rhesus macaque Peripheral Mononuclear Cells (PBMCs) were isolated fromfreshly drawn heparinized blood by Ficoll density gradientcentrifugation. Depletion of CD4+ cells was achieved by magnetic beadseparation using Dynabeads M-450 CD4 (Dynal, Oslo).

Briefly, 96-well polyvinylidine difluoride (PVDF)-backed plates (MAIPNOB 10; Millipore, Bedford, Mass.) were coated with 10 μg anti-humanrIFNγ (R&D Systems Minneapolis, Minn.) per well in 100 μl of PBS at 4°C. for 16-20 hours. Plates were washed three times with PBS, and thenblocked with RPMI-1640 medium containing 10% heat-inactivated FBS. Cellswere cultured at 5×10⁵ per well in 0.1 mL of medium for restimulationwith pools of 20mer peptides comprising the entire amino acid sequenceof HPV16E1, E2, or L1 or matching DMSO concentration in media as anegative control. After 20-24 hr incubation at 37° C., the plates werewashed 6 times with PBS containing 0.005% Tween 20. Plates were thenincubated with 1 μg biotinylated anti-human rIFN-γ (R&D Systems) perwell in 50 μl of PBS-Tween+5% FCS at 4° C. for 16-20 hours. The plateswere washed 6 times with PBS-Tween before the addition 100 μl per wellof Streptavidin-AP conjugate (BD Pharmingen), diluted 1:2000 inPBS-Tween+5% FCS. After 3 washes with PBS-Tween and 3 washes with PBS,spots were developed with one-step NBT/BCIP reagent (Pierce). Spots werecounted using a stereomicroscope.

EXAMPLE 10

Protection of Beagle Dogs from Canine Oral Papillomas Using RecombinantAdenovirus Constructs Expressing COPV E Proteins

Groups of 4-10 beagle dogs were immunized twice s.c. with 10¹¹ vp perdose at Day 0 and Day 30 with recombinant adenoviruses expressing COPV Eproteins or HPV16 L1 as a negative control. Dogs were challenged byscarification at Day 60 at 10 sites of the buccal mucosa. Dogs weremonitored weekly for formation of warts at the challenged sites for 16weeks.

Three experiments were performed: In the first experiment 6 dogs pergroup were immunized with adenovirus constructs expressing E1+E2, orE4+E7, or E1+E2+E4+E7 and 6 dogs were immunized with an adenoviruscontrol expressing HPV16 L1 (4 groups total). In the second experiment,5 dogs per group were immunized with recombinant adenoviruses expressingE1+E2, or E1 alone, or E2 alone, and 4 dogs were immunized with control.In the third experiment, 4 dogs per group were immunized withrecombinant adenoviruses expressing E1 or E2 alone, or the controlvaccine.

The immunization with COPV E2+E1 adenoviruses almost completelyabolished wart formation and greatly reduced the persistence of warts,which appeared. The COPV E2 construct by itself was just as efficaciousas the E1+E2 constructs, while the E1 construct by itself initiallyappeared not to be as potent in reducing disease (Exp. 2) but in arepeat study (Exp. 3) was just as efficacious as the E1+E2 constructs.Also the E4+E7 recombinant adenoviruses were not as potent as the E2 orE1+E2 adenoviruses. Results are shown in FIG. 8.

1. A method of preventing or treating a disease caused by apapillomavirus comprising administering to a mammal a vaccine vectorcomprising a papillomavirus E gene.
 2. A method according to claim 1wherein the mammal is human.
 3. A method according to claim 1 whereinthe vector is an adenovirus vector or a plasmid vector, and the genesare from a human papillomavirus (HPV) serotype which is associated witha human disease state.
 4. A method according to claim 1 wherein theprotein is selected from the group consisting of: E1, E2, E4, E5, E6 andE7 proteins, mutants, and combinations thereof.
 5. A method according toclaim 4 wherein the protein is E1 or E2 protein.
 6. A method accordingto claim 5 wherein the polynucleotide encoding the E protein iscodon-optimized for expression in the recipient's cells.
 7. A methodaccording to claim 1 wherein the vector is an adenoviral vectorcomprising an adenoviral genome with a deletion in the adenovirus E1region, and an insert in the adenovirus E1 region, wherein the insertcomprises an expression cassette comprising: a) a polynucleotideencoding a papillomavirus protein selected from the group consisting ofE1, E2, E4, E5, E6, E7, and combinations thereof, or mutant formsthereof, wherein the polynucleotide is codon-optimized for expression ina human host cell; and b) a promoter operably linked to thepolynucleotide.
 8. A method according to claim 1 wherein the vector is ashuttle plasmid vector comprising a plasmid portion and an adenoviralportion, the adenoviral portion comprising: an adenoviral genome with adeletion in the adenovirus E1 region, and an insert in the adenovirus E1region, wherein the insert comprises an expression cassette comprising:a) a polynucleotide encoding an E protein selected from the groupconsisting of—E1, E2, E4, E5, E6, E7, and combinations thereof, ormutant forms thereof, wherein the polynucleotide is codon-optimized forexpression in a mammalian host cell; and b) a promoter operably linkedto the polynucleotide.
 9. A method according to claim 1 wherein thevector is a plasmid vaccine vector, which comprises a plasmid portionand an expressible cassette comprising a) a polynucleotide encoding an Eprotein selected from the group consisting of E1, E2, E4, E5, E6, E7 andcombinations thereof, or mutant forms thereof, wherein thepolynucleotide is codon-optimized for expression in a mammalian hostcell; and b) a promoter operably linked to the polynucleotide. 10-18.(canceled)
 19. A synthetic polynucleotide comprising a sequence encodinga canine papillomavirus (COPV) protein, or a mutated form of a COPVprotein, the polynucleotide sequence comprising codons optimized forexpression in a human host.
 20. A polynucleotide according to claim 19wherein the protein is selected from the group consisting of; E1, E2,E3, E4, E5, E6, E7, mutants thereof and combinations thereof.
 21. Apolynucleotide according to claim 20 which is selected from the groupconsisting of E1, E2, E4+E7, and E1+E2+E4+E7.
 22. A polynucleotideaccording to claim 19 which is DNA.
 23. A polynucleotide according toclaim 22 which is selected from the group consisting of SEQ.ID.NO. 1,SEQ.ID.NO. 2, SEQ.ID.NO. 3, SEQ.ID.NO. 4, and combinations thereof. 24.An adenovirus vaccine vector comprising and adenoviral genome with adeletion in the E1 region, and an insert in the E1 region, wherein theinsert comprises an expression cassette comprising: a) a polynucleotideencoding a COPV protein selected from the group consisting of E1, E2,E3, E4, E5, E6, E7, mutants thereof, and combinations thereof, whereinthe polynucleotide is codon optimized for expression in a host cell; andb) a promoter operably linked to the polynucleotide.
 25. An adenovirusvector according to claim 24 which is an Ad 5 vector.
 26. A vaccineplasmid comprising a plasmid portion and an expression cassette portion,the expression cassette portion comprising: a) a polynucleotide encodinga COPV protein selected from the group consisting of E1, E2, E3, E4, E5,E6, E7, mutants thereof, and combinations thereof, wherein thepolynucleotide is codon optimized for expression in a host cell; and b)a promoter operably linked to the polynucleotide.
 27. method ofprotecting a mammal from or treating a mammal with a papillomavirusdisease comprising: a) introducing into the mammal a first vectorcomprising: i) a polynucleotide encoding an HPV or COPV protein selectedfrom the group consisting of E1, E2, E3, E4, E5, E6, E7, mutantsthereof, and combinations thereof, wherein the polynucleotide is codonoptimized for expression in a host cell; and ii) a promoter operablylinked to the polynucleotide; b) allowing a predetermined amount o timeto pass; and c) introducing into the mammal a second vector comprising:i) a polynucleotide encoding an HPV or COPV protein selected from thegroup consisting of E1, E2, E3, E4, E5, E6, E7, mutants thereof, andcombinations thereof, wherein the polynucleotide is codon optimized forexpression in a host cell; and ii) a promoter operably linked to thepolynucleotide.
 28. A method according to claim 27 wherein the firstvector is a plasmid and the second vector is an adenovirus vector.
 29. Amethod according to claim 28 wherein the first vector is an adenovirusvector and the second vector is a plasmid. 30-32. (canceled)